Two novel cytochalasins, chaetoconvosins A and B, isolated from Chaetomium convolutum were investigated using electrospray quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS) in positive-ion mode. The main product ions in the high mass range for chaetoconvosins A and B are formed with the loss of H (2) O, CO, or both. The neutral loss of CH (3) NCO can be detected for chaetoconvosin B. However, their fragmentation patterns producing product ions in the low mass range vary greatly. It was found that hydroxyl or H atom linked to C1 is the key leading to the fragmentation differences. McLafferty-type rearrangement (producing the characteristic product ion at m/z 155), four-centered H rearrangement (generating m/z 193), and RDA reaction (producing m/z 203) are directly or indirectly related to the groups linked to C1. The identity of major product ions was supported by a D-labeling experiment.